An important part in unraveling the causes of cancer will be a detailed understanding of how mitogen-triggered signal transduction is integrated into the molecular mechanisms that govern the cell cycle. A large body of evidence indicates that the product of the c-myc gene is a transcription factor involved in these regulatory processes. Our knowledge of the role played by Myc is fragmentary in three important areas: 1) the mechanisms that regulate expression of the c- myc gene: 2) the downstream targets of Myc activity; and, 3) the mechanisms by which Myc regulates its targets. The goal of this proposal is a comprehensive investigation of signaling pathways both upstream and downstream of Myc. Emphasis is place on genetic experiments to understand how these regulatory processes operate in vivo. The first specific aim is to mutate promoter elements directly in a chromosomal c-myc gene. This will be accomplished using targeted homologous recombination, and will allow the in vivo study of specific regulatory circuits in the regulation of the c-myc gene. The second specific aim is to identify downstream Myc target genes, and to determine how Myc regulates their expression. This will be accomplished by testing the expression of specific putative targets in cells devoid of Myc activity (Myc- cells). A second approach will be to use a general screening method, differential display, to search for novel targets whose expression is altered in Myc- cells. Follow up experiments will identify the cis-acting elements in the targets genes that mediate the action of Myc. These experiments will include an investigating of how Myc regulates the expression of cyclins E and A, which have been implicated as downstream targets. The third specific aim is to disrupt the cyclin E gene, which will be accomplished by gene targeting. The comparison of Myc- and cyclin E- phenotypes will reveal which aspects of the growth regulatory activity of Myc are mediated by cyclin E. Experiments to further improve and generalize gene targeting methodologies are also a part of this specific aim. All the proposed experiments are founded on previous accomplishments of this laboratory: 1) the development of a new and efficient gene targeting strategy; 2) the isolation of Myc- cell lines; and, 3) the identification of cyclins E and A as Myc targets. Taken together, the experiments outlined in this proposal are aimed at providing a better understanding of the function of Myc in normal cellular physiology, and ultimately, its oncogenic action.